Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcription is a key aspect of the retroviral life cycle. The enzyme reverse transcriptase requires divalent cations, manganese or magnesium, for function. In some cation-dependent systems substitution of a physiological metal by a nonphysiological metal has been shown to work. We investigated the effect of different cations on HIV reverse transcriptase activity. The studies established reaction conditions for assaying different cations. A variety of transition metals were used in in vitro assays with HIV recombinant RT homodimer and some were delivered to HIV-infected cells in vitro to study effects on virus production. Most metals substituted adequately for magnesium. However, palladium showed a marked nonreversible inhibition of RT activity in vitro that correlated with reduced HIV virus production in tissue culture. A more extensive range of transition metals and divalent cations was tested for their effects on detection of HIV RT from infected cell supernatants. In these complex phenotypes were seen. In some cases the RT activity appeared to be more easily detectable. This may relate to calcium-dependent nucleases in cell supernatants being inhibited, leading to an apparent enhancement of RT activity, or may be due to direct effects on RT processivity.
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PMID:Effects of cation substitutions on reverse transcriptase and on human immunodeficiency virus production. 907 28

Antioxidant enzymes from S. mansoni, cytosolic Cu-Zn superoxide dismutase (CT-SOD), signal-peptide-containing SOD (SP-SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) were compared for their relative levels of transcript expression throughout development in a semiquantitative reverse transcriptase-polymerase chain reaction assay. All of the antioxidant enzymes exhibited a similar pattern of developmental regulation. Adult worms have the highest level of specific mRNA compared with larval stages. GST shows the highest level of expression, being approximately 10-fold more abundant than CT-SOD and SP-SOD and 100-fold more abundant than GPX. This order of expression was nearly consistent for all the developmental stages studied. To localize the antioxidant enzymes, immunofluorescence staining was performed on 3-hr schistosomula and adult worms. GPX, SP-SOD, and CT-SOD were all found to be associated with the adult tegument and gut epithelium. SP-SOD was also associated with organelle and cell membranes of parenchymal cells and interestingly with the spines of adult worms. Schistosomula, on the other hand, showed little immunofluorescence. These studies further demonstrate the developmental regulation of antioxidant enzymes and localize them to the host-parasite interface, supporting the notion that they have a role in allowing adult worms to evade immune attack.
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PMID:Schistosoma mansoni: the developmental regulation and immunolocalization of antioxidant enzymes. 914 42

Using a reverse transcriptase polymerase chain reaction (RT-PCR), a complement DNA encoding secreted superoxide dismutase (s-SOD) of a mouse kidney has been isolated and the nucleotide sequence was determined. The deduced amino acid sequence of mouse s-SOD cDNA shares 79% identity with the rat seminal SOD sequence and 61% identity with the human SOD3 sequence. Northern blot analysis showed that mouse s-SOD is intensely expressed in the kidney and lung tissues and detectable in other tested tissues, including the brain. The mouse s-SOD gene was assigned to chromosome 5 using fluorescence in situ hybridization analysis and PCR analysis of mouse/hamster hybrid cells.
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PMID:Sequence analysis, tissue expression and chromosomal localization of a mouse secreted superoxide dismutase gene. 916 33

The gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of a wide variety of suppurative infections of cutaneous tissues. Previous analyses have demonstrated that the M protein of S. pyogenes is an adhesin that directs the attachment of the streptococcus to keratinocytes in the skin. In this study, we have examined keratinocyte function in response to S. pyogenes and found that adherent versus nonadherent streptococci promote distinct patterns of expression of several proinflammatory molecules and keratinocyte cell fate. When analyzed by a quantitative reverse transcriptase PCR method, infection of cultured HaCaT keratinocytes with adherent, but not nonadherent, streptococci resulted in increased expression of mRNA for the cytokines interleukin-1alpha (IL-1alpha), IL-1beta, and IL-8 but neither infection induced expression of tumor necrosis factor alpha. In contrast, both adherent and nonadherent S. pyogenes induced expression of IL-6 and each promoted synthesis and release of prostaglandin E2 (PGE2). However, considerably greater levels of IL-6 expression were stimulated by adherent streptococci relative to nonadherent streptococci and the kinetics of PGE2 release in response to nonadherent streptococci was delayed compared to the response to adherent streptococci. Staining with the fluorescent probe ethidium homodimer-1 revealed that keratinocyte membranes were rapidly damaged upon infection with adherent streptococci but were not damaged by nonadherent streptococci. Finally, treatments which inhibited streptococcal metabolism completely blocked the ability of adherent streptococci to elicit responses. These data suggest that expression of an adhesin is a strategy used by S. pyogenes to modulate keratinocyte responses during infection of the skin and implicate additional streptococcal products in these signaling interactions.
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PMID:Keratinocyte proinflammatory responses to adherent and nonadherent group A streptococci. 916 41

We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.
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PMID:Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase. 917 79

In contrast to a mutant adhesin-deficient Streptococcus pyogenes (group A streptococcus), its isogenic parental strain binds to human keratinocytes and promotes a vigorous proinflammatory response, characterized by enhanced expression of several cytokines, a more rapid release of prostaglandin E2 (PGE2) and damage to keratinocyte membranes. However, adherence alone is not sufficient to induce these responses. In this study, we have begun to examine the contribution of other streptococcal products in interactions with keratinocytes by the construction and evaluation of mutants deficient in expression of the secreted pore-forming haemolysin, streptolysin O (SLO). Inactivation of SLO did not prevent the streptococci from adhering to cultured HaCaT keratinocytes or from expressing an unrelated second streptococcal haemolysin, streptolysin S, during infection of keratinocytes. As measured by a quantitative reverse transcriptase polymerase chain reaction (PCR) assay, inactivation of SLO also did not have a marked effect on the expression of interleukin 1alpha (IL-1alpha) during infection. However, the lack of the ability to produce SLO was associated with a considerable reduction in expression of IL-1beta, IL-6 and IL-8 by infected keratinocytes. Measurement of the release of PGE2 by an enzyme-linked immunosorbent assay demonstrated that the SLO-deficient mutants were also not capable of promoting the rapid high level of PGE2 release characteristic of the adherent SLO-producing parental strain. Finally, analyses using the fluorescent probe ethidium homodimer-1 and measurements of release of keratinocyte lactate dehydrogenase indicated that the failure of the SLO-deficient mutants to induce responses was associated with the failure of these mutants to damage the integrity of the keratinocyte membrane. These data implicate SLO as a factor that acts synergistically with an adhesin to modulate the signalling responses of keratinocytes during infection.
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PMID:Streptolysin O and adherence synergistically modulate proinflammatory responses of keratinocytes to group A streptococci. 948 89

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.
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PMID:p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors. 949 22

The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding
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PMID:Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up. 1010 27

Transforming growth factor (TGF)-beta1 induces extracellular matrix deposition and proliferation of mesenchymal cells. We recently reported that interleukin (IL)-6 is an essential mediator of growth factor-induced proliferation of lung fibroblasts. Here, we demonstrate by reverse transcriptase polymerase chain reaction and enzyme-linked immunoassay that TGF-beta1 is a potent inducer of IL-6 mRNA and protein in primary human lung fibroblasts. Transient transfections of fibroblasts with a luciferase reporter gene construct containing nucleotides -651 to +1 of the human IL-6 promoter revealed that TGF-beta1 also potently activated IL-6 promoter activity. Progressive 5'-deletions and site-directed mutagenesis of the parental construct located the TGF-beta1-responsive cis-regulatory element to a known activating protein-1 (AP-1) sequence (nucleotides -284 to -276). Gel shift analyses revealed that AP-1 DNA binding activity in nuclear extracts was increased 30 min after stimulation with TGF-beta1. In contrast, neither CCAAT enhancer-binding protein-beta, NF-kappaB, nor Sp1 were activated by TGF-beta1. Supershift analyses demonstrated that the AP-1 complex induced by TGF-beta1 was composed of Jun isoforms and absent of Fos isoforms. Moreover, this complex was found to be a JunD homodimer. Our data thus demonstrate that TGF-beta1 is a potent inducer of IL-6 in primary human lung fibroblasts. The TGF-beta1-activated JunD homodimer may be essential for a majority of the biological effects induced by TGF-beta1 in this cell type, such as proliferation and extracellular matrix synthesis.
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PMID:Transforming growth factor-beta1 induces interleukin-6 expression via activating protein-1 consisting of JunD homodimers in primary human lung fibroblasts. 1021 84

Copper zinc superoxide dismutase (CuZnSOD) is an important enzyme for the detoxification of reactive oxygen species. Particularly in the central nervous system (CNS), reactive oxygen species are often associated with acute brain injuries and chronic neurodegeneration. It has been demonstrated in vivo that there is an inverse correlation between CuZnSOD activity and neuronal death after acute brain injury. To further understand the protective role of CuZnSOD upon neurons, we have generated transgenic mouse lines with targeted expression of the human CuZnSOD gene (SOD1) that is driven by a rat neuron-specific enolase gene promoter in neurons of the CNS. The transgenic SOD1 expression was restricted to the CNS identified by reverse transcriptase polymerase chain reaction and SOD gel electrophoresis assays. The CuZnSOD activity was significantly increased in the brain stem of the transgenic mice. Immunostaining of human CuZnSOD activity showed that Purkinje cells in the cerebellar cortex were the most intensely stained neurons in the CNS of the transgenic mice.
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PMID:Targeted expression of human CuZn superoxide dismutase gene in mouse central nervous system. 1047 83


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